Bacteria and fungi as well as other organisms, including plant pathogens, coexist with all living organisms and besides this fact, pathogenic infections are not frequent because of the efficiency of self defense mechanisms. Microorganisms that invade the human or animal body and plants are challenged by several defense mechanisms.
When host defenses lacks an efficient barrier against pathogen invasion, antibiotics have been used to function as bactericides and, in general, anti-microbials. However, different antibiotics have been continuously sought due to the severe side-effects and the emergence of mutant microorganisms acquired resistance to the long-term used antibiotics. In this regard, attempts to develop novel antibiotics have been carried out by screening secondary metabolites of microorganisms, by synthesizing analogues of known antibiotics such as quinolones or by isolating proteins or peptides induced by intracellular defense mechanisms of plants and animals.
In fact, host defenses include mechanical and chemical factors. One of the chemical defense mechanisms of animals and plants against infection is the production of peptides that have anti-microbial activity. Naturally occurring amphipathic lytic peptides play an important if not critical role as immunological agents and have some defense functions in a range of animals. The function of these peptides is to destroy prokariotic and other non host cells by disrupting the cell membrane and promoting cell lysis. Common features of these naturally occurring peptides include an overall basic charge, a small size (23-39 amino acid residues) and the ability to form amphiphilic α-helices.
Many different families of anti-microbial peptides, classified by their amino acid sequence and secondary structure have been isolated from insects (Steiner, H.; Hltmark, D.; Engstrom, A.; Bennich, H. & Boman, H. G.,1991. Nature.292, 246-248); plants (Cammue, B. P.; De Bolle, M. F.; Terras, F. R.; Proost, P.; Van Danrne, J.; Rees, S. B.; Vanderleyeden, J. and Broekaert, W. F. 1992. J. Biol. Chem. 267. 2228-2233.), mammals (Nicolas, P. & Mor, A. .1995. Annu. Rev. Imunol. 49: 277-304) and microorganisms (Boman, H. G.1995. Annu. Rev. Imunol. 13: 61-92).
Cecropin, cysteine-containing defensin and sapecin, isolated from insects, are examples of antibacterial peptides whose target site is lipid membrane of Gram positive bacteria (Kuzuhara, T. et al. 1990. J. Biochem. 107: 514-518). Studies have demonstrated that Cecropin B isolated from Bombix mori have biological activity against bacterial species (Kadono-Okuda, K. Taniai, K., Kato, Y. Kotani, E. & Yamakawa, M. 1995. J. Invertebr. Pathol. 65, 309-310). Further, it was reported that this peptide when translocated into the intercellular spaces in rice transgenic plants is protected from degradation by plant peptidases and confers enhanced resistance against Xanthomonas oryzae pv. oryzae infection (Sharma, A.;, Sharma, R.; Imamura, M.; Yamakawa, M. & Machii, H. 2000. FEBS. 484: 7-11).
Attacin, sarcotoxin, deftericin, coleoptericin, apidaecin and abaecin are other antibacterial peptides whose target site are lipid membranes. These peptides conserve G and P domains, and have an influence on the cell differentiation of Gram negative bacteria. In particular, attacin has been also reported to break down outer membrane of the targeted bacteria by inhibiting the synthesis of outer membrane proteins.
Besides the above cited antibacterial peptides of insects, several antibiotic peptides have been also isolated from amphibia. Indeed, as other animals, amphibians are rich in anti-microbial peptides (Zasloff, M. 1987. Proc. Natl. Acad. Sci. 89:5449-5453), and many of them belong to the group of amphipathic α-helical structure peptides such as magainins (Daba, H., Pandian, S., Gosselin, J. F. Simard, R. E., Huang, J. and Lacroix, C. 1991. Appli. Environ. Microbiol. 57, 3450-3455), bombinins (Gibson, B. W., Tang, D., Mandrell, R., Kelly, M. and Spindel, E. R. 1991. J. Biol. Chem. 266, 23103-23111), bufonins (Park, C. B., Kim, M. S. and Kim, S. C. (1996) Biochem. Biophys. Res. Comm. 218, 408-413.), dermaseptins (Batista, C. V. C., Silva, L. R., Sebben, A., Scaloni, A., Ferrara, L., Paiva, G. R., Olamendi-Portugal, T., Possani, L. D. and Bloch, C. Jr. 1999. Peptides 20, 679-686) and defensins (Kagan, B. L. et al. 1990. “Anti-microbial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes. Proc Natl Acad Sci. USA. 87(1):210-214). Most of these peptides have been isolated from glands and gastrointestinal tract.
All these molecules has been subject of intense research in order to clarify their biosynthesis, mechanism of action, activity towards microorganisms and potential clinical applications.
An important class of anti-microbial peptides are those known as Magainins. According to Zasloff (1987), at least five proteins may be isolated from the skin of the African clawed frog (Xenopus laevis). The natural proteins are active against a broad range of microorganisms including bacteria, fungi and protozoans. The broad spectrum anti-microbial activity is also present in synthetic peptides and in certain truncated analogs of the natural proteins. Such a class of broad spectrum bio-active polypeptides have been described in the U.S. Pat. No. 5,643,876. These peptides have a molecular weight of about 2500 Da or less, are highly water soluble, amphiphilic and non-hemolytic. They are also defined as a class of substantially pure, homogeneous peptide composed of about 25 amino acids.
The U.S. Pat. No. 5,424,395 discloses a synthetic peptide with 23 amino acid, derived from magainin II showing anti-microbial activity in plants. U.S. Pat. No. 5,912,231 presents a compound comprising a Magainin I or a Magainin II peptide with biological activity, wherein at least one substitution may be made for certain amino acid residues with other amino acids residues. The resulting peptides are known as substitution analogues. Preferred peptides are those obtained by deletion or substitution of at least one amino acid residue in the position 15 andor 23.
U.S. Pat. No. 5,424,395 also describes synthetic peptides derived from Magainin I and Magainin II having anti-microbial activity. The peptides contain 23 amino acid residues and are useful in retarding the growth of plant pathogens.
U.S. Pat. No. 5,912,230 discloses an invention based on substantially pure peptides which have anti-candidal or anti-bacterial activity which are equivalent to that of naturally occurring histatins but are smaller in size. These peptides represent defined portions of the amino acid sequences of naturally occurring human histidine-rich salivary proteins called histatins.
Defensins are relatively small polypeptides of about 3-4 kDa, rich in cysteine and arginine. As a class of anti-microbial peptides, defensins have activity against some bacteria fungi and viruses. The defensins are believed to have molecular conformations stabilized by cysteine bonds, which are essential for biological activity.
The documents U.S. Pat. No. 5,861,378 and U.S. Pat. No. 5,610,139 disclose peptides isolated from horseshoe crab hemocyte, having a similar amino acid sequence to those of defensin and showing strong anti-microbial activities in the fraction 5S, as well as compositions and pharmaceutical preparations using them. They also provide a DNA encoding one or more peptides which show significant physiological activity against Gram positive and Gram negative bacteria and fungi. U.S. Pat. No. 5,610,139 also presents antimicrobial compositions, containing the referred peptides combined with one or more β-lactol or chloramphenicol antibiotics, these compositions exihibing synergistic bactericidal effect against S. aureus infections.
In the U.S. Pat. No. 5,766,624 is proposed a method for treatment of microbe infection in mammals using defensins; U.S. Pat. No. 5,821,224 also presents a β-defensin of 38-42 amino acid, with anti-microbial activity, obtained from bovine neutrophil.
Cathepsin G is a granule protein with chymotripsin-like activity being also known as chymotripsin-like cationic protein. Some polypeptides mutually homologous to cathepsin G are called defensins. In the U.S. Pat. No. 5,798,336 various peptides with anti-microbial activity are provided, being the sequence of said peptides related to amino acid sequences within Cathepsin G. Despite of some of the peptides have showed specificity because of being more effective against determined microorganism, mostly they were effective against both, Gram-negative and Gram-positive bacteria. It is mentioned that pharmaceutical compositions containing these peptides are useful in prophylaxis treatment of infections.
Another type of anti-microbial peptides named buforin was isolated from the stomach tissue of the Asian toad Bufo bufo garugrizans. Two molecules derived from histone H2A were identified, Buforin I and Buforin II which contain 39-aa and 21-aa respectively. These molecules showed different mechanisms of action, having buforin II much stronger anti-microbial activity, killing bacteria without lysing cells and presenting high affinity for DNA and RNA. This suggests that the target of this peptide is the nucleic acids and not the cell membranes (see Park, C. B.; Yi, K.; Matsuzaki, K.; Kim, M. S.; Kim, S. C. 2000. PNAS. 97:8245-8250).
U.S. Pat. No. 5,877,274 provides a novel class of cationic peptides referred to as bactolysins, which have anti-microbial activity and have the ability to significantly reduce the level of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF). In this document, it is also proposed a method of inhibiting either the growth of bacteria or an endotoxernia or sepsis associated disorder by administering a therapeutically effective amount of the peptide.
Each one of these different peptide types is distinguished by sequence and secondary structure characteristics. Based only on the sequence, it is difficult to predict either the activity of a peptide or the secondary structure that it will be formed (Hancock, R. E. W., and Chapple, D. S. 1999. Anti-microbial Agents and Chemotherapy. 43, 1317-1323).
Most of the peptides without disulfide bridges have random structures in water, and when they bind to a membrane or other hydrophobic environment, or, self-aggregate, they form a structure (Bello, J., Bello, H. R., and Granados, E. 1982. Biochemistry 21, 461-465; Falla, T. J., Karunaratne, D. N., and Hancock, R. E. W. 1996. J. Biol. Chem. 271, 19298-19303). For example, cecropins and mellitin only acquire amphiphilic alpha-helices in membranous environments. It is known that the both dual cationic and hydrophobic nature of the peptides is important for the initial interaction between the peptide and that is the cationic character of the bacterial membrane what promotes interaction with bacterial outer and cytoplasmic membranes (Hancock, R. E. W., Falla, T., and Brown, M. H. 1995. Adv. Microb. Physio. 37, 135-175).
Several hypotheses have been suggested for the mechanism of action of the lytic peptides, most of them related to membrane destruction. Whatever the mechanism of lytic peptide-induced membrane damage, an ordered secondary conformation such as an amphiphilic helix and positive charge density are supposed to participate in the peptide-promoted lysis reaction.
Membrane-binding is the first step of the peptide-membrane interaction V mechanism and the knowledge of its determinants and driving force are prerequisites for understanding the mechanism itself and the molecular reasons for the prokaryotic specificity (Saberwal, G., and Nagaraj, R. 1994. Biochem. Biophys. Acta 1197, 109-131). The positively charged peptides were found to bind preferentially to negatively charged membranes what is a major reason for the prokaryotic specificity. The enhanced affinity is caused by an electrostatic attraction of the peptides to the negatively charged membrane surface rather than a specific-lipid interaction (Westerhoff, H. V., Juretic, D., Hendler, R. W., and Zasloff, M. (1989) Proc. Natl. Acad. Sci. USA. 86, 6597-6601).
The present invention discloses a novel class of anti-microbial peptide, isolated from skin of Phyllomedusa hypochondrialis, a kind of frog native to Amazonian, Brazil. It was termed Phylloseptins and its structure did not show any homology with another known peptides.